Nitric oxide (NO), a small molecule which is highly toxic at moderate concentrations, is a key messenger in mammalian physiology. NO is produced in humans by three related enzymes which comprise the nitric oxide synthase (NOS) family.
Endothelial NOS (ENOS) produces NO which controls vascular tone (hence blood pressure), dilates the airways, and controls numerous processes dependent on local dilation of blood vessels (such as gas exchange in lungs, penile erection, and renal function). Brain or neuronal NOS (BNOS or NNOS) produces NO which functions as a neurotransmitter. It controls peristalsis in the gut, and is implicated in neural potentiation and brain development. NNOS and ENOS are constitutive enzymes controlled by intracellular calcium and the regulatory protein calmodulin (CAM). When the level of calcium in the cell rises, NNOS and ENOS bind calmodulin and are turned on to start NO production.
A third, inducible NOS, immune NOS or macrophage NOS (INOS or MNOS), is synthesized by the immune system in response to an immune challenge. Upon induced expression, this enzyme is always active; it has a calmodulin binding site, but binds calmodulin tightly even at low calcium levels. INOS produces orders of magnitude more NO than other NO synthases. This NO level is cytotoxic to tumor cells, bacteria, and other pathogenic organisms.
While INOS thus appears to be an important component of immune response, its activity is highly toxic as well. Excess production of NO by INOS can be responsible for toxic shock syndrome, septic shock, and killing of islet cells in diabetes. Excess NO production by INOS has also been implicated in a wide range of other autoimmune conditions, including arthritis and other inflammatory conditions.
Thus, it is of critical importance to learn to control NO synthesis by one NOS, without interfering with the activity of other NO synthases. Currently, inhibitors of INOS also inhibit NNOS and ENOS.
The current invention concerns recently discovered intrinsic control site elements of constitutive nitric oxide synthases. These intrinsic control site elements, referred to as xe2x80x9cregulatory peptides,xe2x80x9d include the regulatory peptide of endothelial nitric oxide synthase (ENOS), MSGPYNSSPRPEQHKSYKIRFNSVSCSDPLVSSWRRKRKESSNTD (SEQ. ID. NO. 1); the regulatory peptide of neuronal nitric oxide synthase (NNOS) MRHPNSVQEERKSYKVRFNSVSSYSDSRKSSGDGPDLLRDNFE (SEQ. ID. NO. 2); a polypeptide specific to inducible nitric acid synthase (INOS), amino acids 600-615 of INOS (SEQ. ID. NO. 3). Based on this discovery, methods are now available to identify agents that modulate (activate or inhibit) NOS activity, as well as the agents themselves. Agents include agents that inhibit NOS activity by blocking calmodulin activation of the NOS enzyme; agents that inhibit NOS activity by blocking electron transfer from NADPH to an active site in NOS; agents that activate a constitutive NOS enzyme by antagonizing autoinhibition of a regulatory region of the NOS enzyme; and agents that modulate NOS activity by interacting with the regulatory peptide or spatially adjacent control regions. The agents include the peptides described above, as well as derivatives of these peptides, and homologous peptides. Homologous peptides include substantially isolated peptides having an array of at least two positively charged amino acids, and an amino acid sequence of at least about 60% homology, or about 67% homology, or about 80% homology, or about 90% homology, to the amino acid sequence of the ENOS regulatory peptide; peptides having an amino acid sequence of at least about 60% homology, or about 67% homology, or about 80% homology, or about 90% homology, to the amino acid sequence of the NNOS regulatory peptide; peptides having an amino acid sequence of at least about 60% homology, or about 67% homology, or about 80% homology, or about 90% homology, to the amino acid sequence of the INOS-specific peptide; and peptides having an amino acid sequence of at least about 60% homology, or about 67% homology, or about 80% homology, or about 90% homology, to the amino acid sequence of the negatively charged loops of the NOS enzymes. The invention further concerns nucleic acids encoding the peptides, derivatives, and homologous peptides; fusions of peptides with proteins or other macromolecules (e.g., polysacharides); peptidomimetics of the peptides, derivatives, and homologous peptides; and antibodies (either monoclonal or polyclonal antibodies, or fragments thereof) to the peptides, derivatives, and homologous peptides.
The agents can be used to modulate the activity of NOS enzymes. Preferably, the agents modulate the activity of one NOS enzyme, but do not substantially affect the activity of the other NOS enzymes. Such agents can be used to treat diseases or conditions mediated by production of nitric oxide by inducible nitric oxide synthase, such as toxic shock, septic shock, autoimmune diseases, inflammatory conditions, and diabetes. The agents can also be used to treat diseases or conditions mediated by production of nitric oxide by a constitutive NOS enzyme, such as hypertension, diabetes, or AIDS-related dementia.
Thus, as a result of the discovery described herein, it is now possible, for the first time, to design and/or isolate isoform-specific inhibitors, and also isoform-specific activators, of the different NOS isoforms. This overcomes the limitations of earlier methods, which restricted drug discovery to analogs of substrates, such as arginine, or cofactors, such as tetrahydrobiopterin. Furthermore, the current discovery allows identification of activators of NOS enzymes, which was previously impossible.